COMBINATIONS OF THE ALPHA-HELIX-TURN-ALPHA-HELIX MOTIF OF TETR WITH RESPECTIVE RESIDUES FROM LACI OR 434CRO - DNA RECOGNITION, INDUCER BINDING, AND UREA-DEPENDENT DENATURATION

We constructed 10 different variants of TetR by substituting all or so me of the residues in the alpha-helix-turn-alpha-helix (HTH) operator binding motif with the respective amino acids from LacI or 434Cro. The variants were soluble, negative transdominant over tetR in vivo, and as active as wild-type TetR in tetracycline binding in vitro. The urea -induced denaturation of the 10 variants occurs in single reversible t ransitions, which are centered around 4.3 M urea. Denaturation is conc entration-dependent, supporting a simple two-state mechanism in which the folded dimeric protein is in equilibrium with unfolded monomers. A n analysis according to the two-state model yields a Gibbs free energy of stabilization (at 0 M urea, 25 degrees C) of about 75 kJ/mol, typi cal for dimeric proteins of this size. Even a deletion of 24 residues from the reading head decreased the stability by only 2.7 kJ/mol. Thes e results suggest that the DNA reading head of Tet repressor is a ther modynamically independent domain and that the thermodynamic stability of the Tet repressor dimer is determined by the association of the dim erization domains of the individual monomers. Variants containing repl acements in the first alpha-helix of HTH did not show any DNA binding activity whatsoever. We attribute this to the alteration of the two N- terminal residues in this alpha-helix. TetR variants were active in no nspecific DNA binding, when either all or only the solvent-exposed res idues in the recognition alpha-helix of HTH were exchanged to the resp ective LacI sequence. Replacement of the same residues by the respecti ve amino acids from 434Cro yielded hybrid proteins that specifically r ecognize tetO in vitro. Taken together, these results establish that t he similarity of operator recognition between 434Cro and TetR is great er than between TetR and LacI and confirm that prediction of the recog nized DNA sequence is not obvious from the sequence of the respective HTH or recognition alpha-helix.